Reversible post-translation modification of proteins is a major mechanism of transmitting cellular signals and regulating protein function. Protein phosphorylation is a common modification that can act as a "switch" of protein function in response to extracellular signals. Most extracellular signals are propagated by a diverse class of protein kinases called MAP (Mitogen activated protein) kinases, which themselves are tightly regulated by phosphorylation. These phosphorylation cascades signal to chromatin to bring about appropriate gene expression. Recent findings now indicate that specific chromatin modifications (primarily on histone proteins) are critical in regulating expression of target genes induced by these pathways. The most studied histone modification is acetylation of lysine side-chains within the amino terminal tails. Also, phosphorylation, methylation and ubiquitinylation of histones are believed to integrate (synergistically or antagonistically) with acetylation to generate a "histone code" necessary for the proper levels of gene expression from altered chromatin structure. Misregulation of components of these pathways results in a variety of diseases, the most prevalent being cancer. In the previous grant period, we determined the molecular mechanisms of protein phosphatases that down-regulate the MAP kinases. In the next grant period we have shifted our focus to investigate the ultimate target of MAP kinase signaling, chromatin modification. It is unclear how chromatin modifying enzymes carry out their function on nucleosomes. The proposed work will investigate the molecular mechanism of nucleosomal acetylation by histone acetyltransferase (HAT) complexes. Here, we will focus on the basic mechanism of the MYST family of HATs (Aim 1), and investigate how HAT enzymes recognize, acetylate and propagate acetylation on nucleosomal arrays (Aim 2). An enzyme-based "histone code" hypothesis will be investigated using semi-synthetic nucleosomal arrays containing site-specific modifications on histone H3 (Aim 3).